Current transformation methods using Agrobacterium or biolistic bombardment have challenges such as random integration, multiple transgene copies, and unpredicted integration sites. Acting alone or combined, these challenges could lead to unpredictable expression or silencing of introduced transgenes. Though homologous recombination can be explored to address these challenges (Iida and Terada, (2005) Plant Mol. Biol. 59:205-219; Wright et al. (2005) Plant J. 44:693-705), site-specific integration (SSI) mediated by DNA recombinase is practically a more promising approach to eliminate random integration of unpredictable copies of a transgene by placing single copy transgene into a pre-characterized site in plant genome.
Several site-specific DNA recombination systems, such as the Cre/Iox of bacteriophage P1, the FLP/FRT of Sacchromyces cerevisiae, and the R/RS of Zygosacchromyces rouxii have been used in site-specific gene integration studies (Groth and Calos, (2003) J. Mol. Biol. 335:667-678; Ow, (2003) Plant Mol. Biol. 48:183-200). A common feature of these systems is that each system consists of a single polypeptide recombinase Cre, FLP, or R, and two identical or almost identical palindromic recognition sites Iox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked on each side by an inverted repeat sequence where the corresponding recombinase specifically binds. If two recognitions sites are located in cis on the same DNA molecule, DNA segment flanked by the two sites can be excised if the two sites are in the same orientation, or be inverted if the two sites are in opposite orientations. If two recognitions sites are each located in trans on two different DNA molecules, a reciprocal translocation can happen between the two linear DNA molecules, or the two molecules can integrate if at least one of them is a circular DNA (Groth and Calos, J. Mol. Biol. 335:667-678 (2003); Ow, Plant Mol. Biol. 48:183-200 (2003)).
A simple SSI can target DNA into single recombination site previously placed in a plant genome. Improvement of the single site integration approach involved transient Cre expression and the use of mutant Iox sites to recreate two less compatible Iox sites after integration to reduce subsequent excision of the integrated gene in tobacco (Albert et al. (1995) Plant J. 7:649-659; Day at al. (2000) Genes Dev. 14:2869-2880). Similar approach was used to produce SSI events in rice by biolistic bombardment transformation method and the transgene was proven to be stable and consistently expressed over generations (Srivastava and Ow, (2001) Mol. Breed. 8:345-350; Srivastava et al. (2004) Plant Biotechnol. J. 2:169-179). Using Agrobacterium T-DNA for donor DNA delivery and a promoter trap to activate selectable marker gene and to displace Cre expression upon DNA recombination, ˜2% single Iox site SSI was achieved in Arabidopsis (Vergunst et al. (1998) Nucleic Acids Res. 26:2729-2734). The process of SSI is basically irreversible and thus the genomic site can not be recovered for repeated use. Additionally, since SSI will integrate the entire circular DNA, unwanted components such as the vector backbone is also integrated unless the integration DNA can be circulated by Cre recombinase to remove unwanted DNA prior to SSI (Srivastava et al. (2004) Plant Biotechnol J. 2:169-179; Chawla et al. (2006) Plant Biotechnol. J. 4:209-218; Vergunst et al. (1998) Nucleic Acids Res. 26:2729-2734). To achieve marker-free site-specific gene integration, a two-step approach was proposed to combine gene integration using one recombinase system such as Cre/Iox followed by gene excision using another system such as FLP/FRT that is also conditionally controlled by an inducible promoter (Srivastava and Ow, (2004) Trends Biotech. 22:627-629).
If two incompatible recognition sites, which are similar enough to be recognized by the same recombinase but also different enough to prevent DNA recombination from happening between them, are located on a linear DNA molecule, DNA segment between the two sites will not be either excised or inverted. When a circular DNA molecule carrying an identical pair of the incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA molecule with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently happen between any pair of compatible sites and result in the restoration of the original two DNA molecules or the exchange of the intervening DNA segments between the two DNA molecules. The latter process termed recombinase mediated cassette exchange (RMCE) can be employed to integrate transgenes directionally into predefined genome sites (Baer and Bode, (2001) Curr. Opin. Biotechnol. 12:473-480; Trinh and Morrision, (2000) J. Immunol. Methods 244:185-193).
RMCE using two identical but oppositely orientated RS sites resulted in donor cassette exchange into the previously placed target site in tobacco (Nanto et al. (2005) Plant Biotechnol. J. 3:203-214). The donor vector containing the R recombinase gene and a third RS site to help eliminating random integration was delivered by Agrobacterium transformation. RMCE utilizing both the Cre/Iox and FLP/FRT systems was used in animal cell cultures to improve RMCE frequency (Lauth et al. (2002) Nucleic Acids Res. 30:e115). RMCE using two directional incompatible FRT sites was used in Drosophila to achieve cassette exchange by transiently expressed FLP recombinase between a target DNA previously placed in the genome and a donor introduced as a circular DNA (Horn and Handler, (2005) Proc. Natl. Acad. Sci. 102:12483-12488). A complex gene conversion approach involving Cre/Iox and FLP/FRT mediated site-specific integration, RMCE, and homologous recombination was explored in maize (Djukanovic et al. (2006) Plant Biotechnol. J. 4:345-357).